1. Field of the Invention
The invention relates generally to chemistry and protein science. More specifically, it concerns methods and compositions for synthesizing polypeptides and proteins, and more particularly, a convergent, kinetically-controlled method and intermediates for covalently assembling multiple peptide fragments into a full length polypeptide.
2. Description of Related Art
In the past decade total chemical synthesis has proved to be a robust and reproducible method for using amino acid sequence data predicted from genome sequencing to make high purity polypeptide chains that fold with great efficiency to give proteins of defined tertiary structure and full biological activity (Dawson and Kent, 2000; Kochendoerfer et al., 2003; Bang and Kent, 2004). This success has been made possible by the introduction of ‘chemical ligation’ methods, based on the chemoselective reaction of unprotected peptides under mild conditions in aqueous solution. Chemical synthesis gives precise atom-by-atom control over the structure of the protein molecule. Chemistry has been used to make analogue protein molecules containing multiple non-coded amino acids, fixed elements of secondary structure, backbone-modified polypeptide chains, or non-linear peptide chain topologies (Dawson and Kent, 2000). By correlating defined changes in the chemical structure of the protein molecule with the effects on folding and function, the inventors can gain new insights into the principles governing the biological activities of proteins.
Recent improvements in synthetic methods have been focused on the preparation of proteins by sequential ligation of three or four peptide segments (Bang and Kent, 2004; Canne et al., 1999; Bang and Kent, 2005, which are incorporated by reference). See also U.S. Pat. No. 6,184,344 (in solution); Canne et al. (1999) and U.S. Pat. No. 6,326,468 (in solid phase); and U.S. Pat. Publication 2005/0113563, all of which are hereby incorporated by reference.
Although syntheses of large proteins have been reported, these are regarded as exceptional ‘tours de force’ (Casi and Hilvert, 2003); in general, synthesis of larger molecules is laborious, time-consuming, and gives low yields, and for these reasons is not widely practiced. Ultimately, there is a need to extend routine total chemical synthesis to proteins more typical of the average size found in nature (approximately 35 kilodaltons, approximately 300 amino acids). The present invention addresses this need.